Fluid exchange methods and devices

ABSTRACT

Methods and devices for exchanging therapeutic agents, such as cells, from one liquid medium to another liquid medium are provided. Aspects of embodiments of the methods include transferring a therapeutic agent from a first medium, such as a freezing, storage or shipping buffer, into a second medium, such as a sterile physiologically compatible buffer. In certain aspects, the transfer of the therapeutic agent from a first medium to a second medium involves the use of acoustic-focusing, or acoustophoresis. Embodiments of the subject methods may facilitate the transfer of a therapeutic agent from a storage medium to an infusion medium, and in certain embodiments include administering the therapeutic agent contained in the infusion medium to the subject. Also provided by the present disclosure are devices for practicing the subject methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application is a divisionalapplication of U.S. patent application Ser. No. 14/237,857, filed Feb.7, 2014, which application is a 371 national phase filing ofInternational Application No. PCT/US2012/057997, filed Sep. 28, 2012,which application claims priority to the filing date of the U.S.Provisional Patent Application Ser. No. 61/541,870, filed Sep. 30, 2011,the disclosures of which are incorporated herein by reference.

INTRODUCTION

High-dose chemotherapy followed by autologous peripheral blood stem cell(PBSC) transplantation is used for many hematological malignancies.PBSCs are often mobilized from the bone marrow with cytokines such asrecombinant G-CSF (rhuG-CSF) and then cryopreserved. This processrequires the addition of cryopreservatives such as dimethyl sulphoxide(DMSO) to prevent cell lysis during freezing. The occurrence of adverseevents during the infusion of previously cryopreserved autologous PBSCis known, and has traditionally been attributed to the presence of DMSOin the thawed cell suspension.

Cryopreserved cell preparations containing DMSO can be washed prior toinfusion. Typically, such cell preparations are washed bycentrifugation-based methods. Centrifugation-based methods havedisadvantages related to batch processing with manual steps that areerror-prone, incomplete fluid exchange, hypoxia in the pelleting of livecells, and incomplete re-suspension of the pelleted particles. Suchprocesses typically require mechanical devices with power requirementsinconvenient for routine bedside use in a clinical setting.

Alternatively, cell preparations can be washed by filtration carried outusing a porous membrane to separate fluid and small particles fromlarger particles, e.g., the cells to be transplanted. Limitations offiltration methods are well known, and include clogging of the membraneas large particles accumulate, which can change the fluid dynamics in apoorly controlled way, limit the purity of the desired particles bytrapping undesired particles as the pore size of the membrane iseffectively changed by accumulation of large particles, and damage thelarge particles by aggregating them under increasing fluid pressure andshear and hypoxia associated with clumping of living cells. The largeparticles can be difficult to recover and the process is usuallyrestricted to batch processing with incomplete fluid transfer.

SUMMARY

Methods and devices for exchanging therapeutic agents, such as cells,from one liquid medium to another liquid medium are provided. Aspects ofembodiments of the methods include transferring a therapeutic agent froma first medium, such as a freezing, storage or shipping buffer, into asecond medium, such as a sterile physiologically compatible buffer. Incertain aspects, the transfer of the therapeutic agent from a firstmedium to a second medium involves the use of acoustic-focusing, oracoustophoresis. Embodiments of the subject methods facilitate thetransfer of a therapeutic agent from a storage medium to an infusionmedium, and in certain embodiments include administering the therapeuticagent contained in the infusion medium to a subject. Also provided aredevices and kits that find use in practicing embodiments of the methods,e.g., as described below.

In certain embodiments, methods of the present disclosure involve theuse of acoustophoresis to transfer a therapeutic agent from a firstmedium, such as a freezing, storage or shipping buffer, into a secondmedium. Such acoustophoresis may include the use of one or moreacoustophoresis devices, such as 2 or more, including 5 or more, 10 ormore, or 20 or more. A range of acoustophoresis devices may be used inpracticing the subject methods, varying in some embodiments in terms ofscale (e.g., macro- or micro-scale acoustophoresis); chip material(e.g., silicon, glass, etc.); chip dimensions; number of separationchannels (e.g., 1 or more, 2 or more, 5 or more, 10 or more, 20 or more,etc.); orientation of the separation channels (e.g., serial, parallel,and/or both); dimensions of the separation channel(s); number of inputsand outputs; type of vibration generator (e.g., a piezoceramictransducer, such as lead zirconate titanate (PZT)); number of vibrationgenerators; frequency and/or voltage applied to the vibration generator;flow rate (e.g., about 1 μl/min, about 100 μl/min, about 1 ml/min, orabout 100 ml/min or more); presence or absence of pumps or valves (e.g.,one or more syringe pumps, elastomeric pumps, and/or peristaltic pumps);and the like, as shall be described more fully herein. In certainaspects, acoustophoresis includes gravimetric fluid flow, and/ormechanically-assisted fluid flow (e.g., using a pump, such as a syringepump or a peristaltic pump).

Further, acoustophoresis devices of interest include, but are notlimited to, devices as described in U.S. Pat. No. 6,929,750; Laurell, etal. (2007) Chem. Soc. Rev., 2007, 36, 492-506; Petersson, et al. (2005)Analytical Chemistry 77: 1216-1221; and Augustsson, et al. (2009) Lab ona Chip 9: 810-818; the disclosures of which are incorporated herein byreference.

A broad range of therapeutic agents may be transferred from one fluidmedium to another by using methods of the present disclosure. In certainaspects, a therapeutic agent is a cell, such as a peripheral blood stemcell (PBSC), umbilical cord blood cell, hematopoietic stem cell, orinduced pluripotent stem cell. Therapeutic agents of interest furtherinclude, but are not limited to, drugs, nucleic acids, proteintherapeutics (e.g., antibodies, such as monoclonal antibodies; andpeptides), blood and blood products, and chemotherapy agents. In certainembodiments, a therapeutic agent is contained within or coupled to aparticle, carrier, vesicle, or other delivery device, such as a bead ora liposome. Therapeutic agents of interest include agents of biologicalorigin, including agents obtained from an in vivo source (e.g., amammalian subject, a human subject, etc.), as well as agents ofnon-biological origin (e.g., chemical or synthetic origin).

In certain aspects, a therapeutic agent is transferred from one mediumto a different type of medium, such as from a freezing, storage orshipping buffer, into a sterile physiologically compatible buffer.Aspects may also, or instead, include transferring a therapeutic agentfrom a medium into the same type of medium. In certain aspects,transferring the therapeutic agent to a different medium may have theeffect of removing one or more components from the environment in whichthe therapeutic agent is present, such as cryopreservatives (e.g., DMSO,glycerol), density gradient reagents (e.g., Ficoll), enzymes (e.g.,collagenase), lysing reagents (e.g., red cell lysing reagents),antibodies, lipids, etc.

In certain embodiments, the therapeutic agent is administered to asubject, such as by infusion. Administration of a therapeutic agent to asubject may be achieved in various ways, including, but not limited to,oral, parenteral (e.g., subcutaneous, intramuscular, intradermal,intravenous and intrathecal), intraperitoneal, intravesicular, etc.,administration. Subjects suitable for the methods of the presentdisclosure include mammals (e.g., humans). Aspects of the methodsinclude allotransplantation, xenotransplantation, orself-transplantation. In certain aspects, the methods are performedunder sterile conditions and/or are sterile.

Also provided by the present disclosure are devices for practicing thesubject methods. In certain embodiments, devices may include a firstcontainer providing a therapeutic agent suspended in a first medium, asecond container providing a second medium, and an acoustophoresisdevice in fluid communication with the first and second containers andconfigured to transfer the therapeutic agent from the first medium intothe second medium.

Devices of the present disclosure may include one or more processorsconfigured to control the device. In certain aspects, a processor may beconfigured to control an acoustophoresis device, such as by altering oneor more of the flow rare (e.g., by controlling one or more pumps), theshape, frequency end/or power of the electrical energy delivered to thevibration generator. Aspects of the present disclosure further includeclosed-loop devices.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 provides a schematic depiction of a microfluidic acoustophoresisdevice that allows for the transfer of a therapeutic agent (e.g., cells)from a first medium into a second medium by acoustic focusing.

FIG. 2, Panels A-C provide cross-section illustrations of a separationchannel of an acoustophoresis device. Particles begin by flowing alongthe sides of the channel (Panel A). An acoustic standing wave may beinduced in the channel (e.g., using a vibration generator, such as apiezoelectric transducer, placed adjacent to the channel), as indicatedby the dashed lines (Panels B-C). The acoustic standing wave creates apressure node in the center of the channel (Panel B). Particles presentin the channel may move towards the pressure node (Panel C).

FIG. 3 provides a schematic cross-section of an acoustophoresisseparator chip. In this embodiment, the separator chip 100 includes achip 12 in which a channel 10 has been formed, such as by etching. Amembrane 14 (e.g., a glass membrane, such as boron silica glass) isattached to the top of the chip 12. A vibration transducer 20 is affixedto the bottom of the chip 12, using affixing means 18 (e.g., adhesives,gels, and the like).

FIG. 4 provides a schematic depiction of an embodiment of the presentdisclosure. In this embodiment, a gravimetric fluid flow is used to passcells through the acoustophoresis device wherein cells are transferredfrom a first storage medium into a second, sterile physiological medium,labeled as a wash buffer. Alternatively, a pump mechanism, such as aperistaltic pump, can be used to control the flow of the fluid throughthe acoustic-focusing chip and into the patient. All tubing and theacoustophoresis chip can be connected and maintained in an entirelyclosed, aseptic system using connectors and methods known in the art.

FIG. 5 provides a schematic depiction of an embodiment of the presentdisclosure. This embodiment is a variant of the embodiment presented inFIG. 4, wherein cells are passed through a plurality of acoustophoresisdevices.

DETAILED DESCRIPTION

Methods and devices for exchanging therapeutic agents, such as cells,from one liquid medium to another liquid medium, are provided. Aspectsof embodiments of the methods include transferring a therapeutic agentfrom a first medium, such as a freezing, storage or shipping buffer,into a second medium, such as a sterile physiologically compatiblebuffer. In certain aspects, the transfer of the therapeutic agent from afirst medium to a second medium involves the use of acoustophoresis.Embodiments of the subject methods facilitate the transfer of atherapeutic agent from a storage medium to an infusion medium, and incertain embodiments include administering the therapeutic agentcontained in the infusion medium to the subject. Also provided by thepresent disclosure are devices for practicing the subject methods.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Methods

As described above, the present disclosure provides methods forexchanging therapeutic agents, such as cells, from one liquid medium toanother liquid medium. Aspects of embodiments of the methods includetransferring a therapeutic agent from a first medium into a secondmedium. In certain aspects, the transfer of the therapeutic agent from afirst medium to a second medium involves the use of acoustophoresis.Embodiments of the subject methods facilitate the transfer of atherapeutic agent from a storage medium to an infusion medium, and incertain embodiments include administering the therapeutic agentcontained in the infusion medium to the subject.

Various steps and aspects of the methods shall now be described ingreater detail below.

Therapeutic Agents

As used, herein, the term “therapeutic agent” means any agent that mayhave a biological function and/or therapeutic effect in a subject. Forexample, a therapeutic agent may be one or more cells (e.g., stem cells,such as PBSCs, umbilical cord blood cells, hematopoietic stem cells,induced pluripotent stem cells, and the like). Examples of suchtherapeutic agents include, but are not limited to, approved celltherapy products (e.g., U.S. Food and Drug Administration approved celltherapy products) including HemaCord (hematopoietic progenitor cells,cord blood; distributed by New York Blood Center Inc., New York, N.Y.);Provenge (Sipuleucel-T; distributed by Dendreon Corporation, Seattle,Wash.); and Laviv (Azficel-T; distributed by Fibrocell Technologies,Exton, Pa.). Therapeutic agents of interest further include, but are notlimited to, protein therapeutics (e.g., antibodies, such as monoclonalantibodies; and peptides), blood and blood products, and chemotherapyagents.

In some instances, a therapeutic agent to be transferred has a diameterof about 0.25 micron or greater, such as 1 micron or greater, includingabout 1 to 10 microns, about 10 to 20 microns, about 20 to 30 microns,about 30 to 40 microns, about 40 to 50 microns, about 50 to 60 microns,about 60 to 70 microns, about 70 to 80 microns, about 80 to 90 microns,about 90 to 100 microns, about 100 to 125 microns, about 125 to 150microns, or about 150 microns or more.

Further, in some instances the therapeutic agent to be transferred has adensity greater than at least one of the medium in which it is containedand the medium into which it is being transferred. In certain aspects,the therapeutic agent to be transferred has a compressibility greaterthan at least one of the medium in which it is contained and the mediuminto which it is being transferred.

In certain aspects, an isolated therapeutic agent has a size, density,and/or compressibility not amenable to transfer by acoustophoresis andis instead contained within, associated with, or coupled to a particleto facilitate acoustophoresis of the therapeutic agent. In certainaspects, the particle has a size, density, and/or compressibilityamenable to acoustophoresis. Aspects of the subject methods includepreparing a therapeutic agent so that it is contained within, associatedwith, or coupled to a particle to facilitate acoustophoresis of thetherapeutic agent. Any convenient particle amenable to acoustophoresisto which a therapeutic agent may be associated (e.g., covalently and/ornon-covalently, directly and/or indirectly, etc.) may be employed inpracticing the subject methods.

Particles of interest include vesicles, such as liposomes. Liposomesamenable to transfer by acoustophoresis and comprising a therapeuticagent may be made using any convenient method and material. Thetherapeutic agent may be contained in the aqueous interior of theliposome and/or associated with a surface, e.g., interior or exterior,of the liposome. As such, in certain aspects, the therapeutic agent isalso, or instead, contained on the exterior surface of the liposome orwithin the lipid bilayer of the liposome.

In some embodiments, the particle is a bead (e.g., a polymer bead) towhich the therapeutic agent is attached. A therapeutic agent may beattached to a bead directly (e.g., covalently or non-covalently) orindirectly (e.g., with one or more binding intermediates, to which thetherapeutic agent is covalently or non-covalently attached). Methods ofmanufacturing beads and attaching therapeutic agents are known in theart.

Accordingly, a broad range of therapeutic agents may be used inpracticing the subject methods, including drugs, nucleic acids, etc. Incertain aspects, a therapeutic agent is a therapeutic protein, such as atherapeutic protein contained within a liposome. Therapeutic proteins ofinterest include, but are not limited to, proteases, proteaseinhibitors, cytokines, chemokines, gonatotrophins, chemoactins,lipid-binding proteins, pituitary hormones, growth factors,somatomedians, immunoglobulins, interleukins, sex hormone bindingglobulin, interferons, growth hormone releasing hormone, parathyroidhormone, calcitonin, leuprolide, insulin-like growth factor-1 andscaffolding proteins such as DARPINs, knottins, FN3 domains, CH domains,Elastin-like polypeptides, and cyclic polypeptides, such as CYCLOTIDEs.Therapeutic proteins of interest include antibodies (e.g., a humanizedantibody) and antigen fragments thereof.

Therapeutic agents of interest include agents obtained from an in vitrosource (e.g., laboratory cells grown in culture), from an in vivo source(e.g., a mammalian subject, a human subject, etc.), or from anon-biological source (e.g., synthetic and/or chemical source). In someembodiments, a therapeutic agent is obtained from an in vitro source. Invitro sources include, but are not limited to, prokaryotic (e.g.,bacterial, archaeal) cell cultures, environmental samples that containprokaryotic and/or eukaryotic (e.g., mammalian, protest, fungal, etc.)cells, eukaryotic cell cultures (e.g., cultures of established celllines, cultures of known or purchased cell lines, cultures ofimmortalized cell lines, cultures of primary cells, cultures oflaboratory yeast, etc.), tissue cultures, and the like.

In some embodiments, the therapeutic agent is obtained from an in vivosource, including agents obtained from tissues (e.g., a tissue biopsy,cell suspension from a tissue sample, etc.) and/or body fluids (e.g.,whole blood, fractionated blood, plasma, serum, saliva, lymphatic fluid,interstitial fluid, etc.). In some cases, a therapeutic agent derivedfrom a subject is cultured, stored (e.g., cryopreserved), or manipulatedprior to practicing the subject methods.

In certain embodiments the source of the therapeutic agent is a “mammal”or “mammalian”, where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some instances,the source of the therapeutic agent is human.

Media

A therapeutic agent may be contained in a medium. For instance, atherapeutic agent may be contained in suspension at any desiredconcentration. For example, where the therapeutic agent is a cell, themedium can contain 10¹¹ or less, 10¹⁰ or less, 10⁹ or less, 10⁸ or less,10⁷ or less, 10⁶ or less, 10⁵ or less, 10⁴ or less, 10³ or less, 500 orless, 100 or less, 10 or less, or one cell per milliliter. Anyconvenient media may be used in practicing the subject methods,including, but not limited to, freezing buffers, storage buffers,shipping buffers, physiologically compatible buffers, sterile buffers,and the like. In some instances, the medium into which a therapeuticagent is being transferred has a higher density than does the medium inwhich the therapeutic agent is contained.

In certain aspects, a medium may include one or more solutes in additionto the therapeutic agent. For instance, a medium may also include one ormore cryopreservatives (e.g., DMSO, glycerol, propylene glycol,hydroxyethyl starch), density gradient reagents (e.g., Ficoll), enzymes(e.g., collagenase), lysing reagents (e.g., red cell lysing reagents),antibodies, lipids, etc. In other aspects, the medium may contain nosolutes other than the therapeutic agent.

Embodiments of the subject methods include media containing two or moredifferent therapeutic agents, including 3 or more, such as 4 or more, 5or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. Thedifferent therapeutic agents may differ in any respect from one another,such as the type (e.g., cell vs. protein therapeutic), sub-type (e.g.,PBMCs vs. umbilical cord blood cells), etc.

Aspects of embodiments of the subject methods include transferring atherapeutic agent from one medium (e.g., a first medium) into anothermedium (e.g., a second medium). In certain aspects, a therapeutic agentmay be subsequently transferred from the second medium to a third,fourth, fifth, etc., medium. For instance, in certain embodiments atherapeutic agent may be transferred from a first medium into a secondmedium, and subsequently transferred from the second medium into a thirdmedium, and subsequently transferred from the third medium to a fourthmedium, etc.

The medium into which a therapeutic agent is transferred may be the sametype or a different type of medium compared with the medium from whichthe therapeutic agent is being transferred. In certain aspects, themedium is the same type (e.g., both the first medium and the secondmedium are physiologically compatible buffers). In other aspects, themedia are of different types, such as the first medium is a freezingbuffer and the second (or third, fourth, etc.) medium is aphysiologically compatible buffer. Any convenient media into which atherapeutic agent may be transferred may be used.

In certain aspects, the first medium is a freezing buffer. In certainaspects, a freezing buffer is a medium containing one or morecryoprotective agents, such as DMSO, glycerol, propylene glycol, etc.The freezing buffer may facilitate the freezing (e.g., cryopreservation)of a therapeutic agent, such as cells. In certain aspects, the freezingbuffer may be thawed prior to or in conjunction with practicing thesubject methods, using any convenient thawing method known in the art.Examples of freezing buffers of interest include, but are not limitedto, Recovery™ cell culture freezing medium (Life TechnologiesCorporation, Carlsbad, Calif.); Synth-a-Freeze® cryopreservation medium(Life Technologies Corporation, Carlsbad, Calif.); CPZ™ cryopreservationmedia (INCELL Corporation, San Antonio, Tex.); EZ-CPZ™ cryopreservationmedia (INCELL Corporation, San Antonio, Tex.); Cell Freezing Medium-Ifreezing buffer (Atlanta Biologicals, Lawrenceville, Ga.); Cell FreezingMedium-II freezing buffer (Atlanta Biologicals, Lawrenceville, Ga.); andCryoStor® cell cryopreservation media (Sigma-Aldrich, St. Louis, Mo.).

Embodiments include a first medium having a concentration ofcryoprotectant of 1% (v/v) or more, including about 2% (v/v) to about 5%(v/v), about 2% (v/v) to about 5% (v/v), about 5% (v/v) to about 7.5%(v/v), about 7.5% (v/v) to about 10% (v/v), about 10% (v/v) to about 15%(v/v), about 20% (v/v) to about 25% (v/v), or about 25% (v/v) to about30% (v/v). Two or more cryoprotectants, such as 3 or more, including 4or more, 5 or more, 6 or more, or 7 to 10, may be present in a firstmedia. Where two or more cryoprotectants are present in a medium, thecryoprotectants may be present at the same concentration (e.g., 5% DMSOand 5% HES) or different concentrations (e.g., 5% DMSO and 6% HES).

In certain embodiments, a buffer may be contained within a container,such as a cryocontainer. Containers of interest include containers thatare Ethinyl Vinyl Acetate (EVA) based, and containers that are not EVAbased (e.g., Teflon, Kaplon, FEP/Polyimide, stainless steel, and thelike). In certain aspects, the container is an EVA freezing bag, such asa Cryocyte™ freezing bag (Baxter Healthcare Corporation, Deerfield,Ill.), Cell-Freeze® cryogenic freezing bag (Charter Medical,Winston-Salem, N.C.), OriGen Cryostore™ freezing bag (OriGen BioMedical,Austin, Tex.), and the like.

Embodiments of the subject methods include transferring a therapeuticagent to a physiologically compatible buffer. Examples ofphysiologically compatible buffers of interest include, but are notlimited to, electrolyte solutions approved for infusion and/or injectioninto human subjects (e.g., U.S. Food and Drug Administration approvedelectrolyte injection solutions). In certain aspects, thephysiologically compatible buffer is sterile. Examples ofphysiologically compatible buffers of interest include, but are notlimited to, dextrose monohydrate injection solution (e.g., 5% DextroseInjection, such as that distributed by Hospira Inc., Lake Forest, Ill.),Lactated Ringer's Injection solution (Hospira Inc., Lake Forest, Ill.),PLASMA-LYTE solutions (Baxter Healthcare Corporation, Deerfield, Ill.);and Isolyte® S solutions (B. Braun Medical Inc., Ontario, Canada).

Acoustophoresis Devices

Transferring a therapeutic agent from one medium into another medium mayinclude the use of an acoustophoresis device. As used herein, the terms“acoustic-focusing device” and “acoustophoresis device” are used broadlyand generically to refer to a device in which particulate matter in afluid may be controlled or manipulated by means of ultrasonic standingwaves, and the terms may be used interchangeably. Examples ofacoustophoresis devices of interest include, but are not limited to,those described in U.S. Pat. No. 6,929,750; Laurell, et al. (2007) Chem.Soc. Rev., 2007, 36, 492-506; Petersson, et al. (2005) AnalyticalChemistry 77: 1216-1221; and Augustsson, et al. (2009) Lab on a Chip 9:810-818; the disclosures of which are incorporated herein by reference.

FIG. 1 a schematic depiction of a microfluidic acoustophoresis devicethat allows for the transfer of a therapeutic agent from a first mediuminto a second medium by acoustic focusing. In this example, thedirection of fluid flow is from the top to the bottom of the figure. Theacoustophoresis device includes two sample inlets and a buffer inlet.With the inputs arranged as illustrated, the sample fluid (dark gray;corresponding to a first medium comprising a therapeutic agent) flowsalong the sides of the channel, with the buffer (light gray;corresponding to a second medium) flowing between, with the fluidsoperating under laminar flow. As such, the first liquid medium and thesecond liquid medium are combined in a manner sufficient to produce alaminar flow of the first and second media, i.e., a flow in which thetwo media are flowing in distinct but adjacent and contacting flowpaths. The densities of the first and second media differ in someinstances in order to ensure the production of the laminar flow uponcombination, where in some instances the density difference between thefirst and second media is 1% or greater, such as 5% or greater,including 10% or greater. A piezoelectric transducer is located belowthe channel which, when activated, creates an acoustic standing wave inthe channel. The acoustic standing wave causes certain particlescontained in the samples to move from the sides of the channel in thefirst media towards the pressure node formed in the center of thechannel (as indicated by the focusing zone; top inset) in the secondmedia. These particles (e.g., the therapeutic agent), now contained inbuffer (that is, the second medium), are collected by the washed sampleoutlet. Two outlets placed at the sides of the channel collect waste.

General principles of certain aspects of acoustophoresis devices areillustrated in FIG. 2, Panels A-C, which provide cross-sectionillustrations of an acoustophoresis separator channel. As depicted inthese panels, particles begin by flowing along the sides of the channel(FIG. 2, Panel A). An acoustic standing wave may be induced in thechannel (e.g., using a vibration generator, such as a piezoelectrictransducer, placed adjacent to the channel), as indicated by the dashedlines (FIG. 2, Panels B-C). The acoustic standing wave creates apressure node in the center of the channel (FIG. 2, Panel B). Certainparticles present in the channel may move towards the pressure node(FIG. 2, Panel C), depending upon their physical properties. Generally,molecules and particles smaller than about 1 micron in diameter are notaffected by the acoustic standing wave(s).

The mechanism by which acoustophoresis devices operate is described in,for example, Laurell, et al. (2007) Chem. Soc. Rev., 2007, 36, 492-506.Briefly, an acoustic contrast factor (also called a Φ-factor) depends onboth a particle's (e.g., a cell) density (ρ_(c)) and its compressibility(β_(c)) in relation to the corresponding properties of the surroundingmedium (ρ_(w), β_(w)). An acoustic contrast factor may be positive ornegative, which determines the direction of the acoustic force andwhether a particular particle will move towards a standing pressure wavenode (i.e., the center of the image in FIG. 2, Panel B) or towards thepressure antinode (i.e., the sides of the channel in FIG. 2, Panel B).Generally, solid particles in aqueous media are moved towards a pressurenode. Accordingly, depending on the application, the shape anddimensions of the channel(s), the materials from which theacoustophoresis device channel is made, the number of inlets and outletsemployed, the flow rate in the channel, the frequency of ultrasoundapplied, and other parameters of an acoustophoresis device may vary.

In certain aspects, an acoustophoresis device may be based upon theLund-method, in which acoustic concentration or separation of suspendedparticles is based on a laminar flow microchannel that is ultrasonicallyactuated from below using a vibration generator, such as a piezoelectricceramic. The width of the channel may be chosen to correspond to halfthe desired ultrasonic wavelength, thereby creating a resonator betweenthe side walls of the flow channel in which a standing wave can beformed. The induced standing wave may thus be generated orthogonal tothe incident ultrasonic wave front. As suspended particles with apositive Φ-factor perfuse the channel they are moved, by means of theaxial primary radiation force (PRF), towards the pressure nodal planealong the channel center, while those with a negative Φ-factor are movedtowards the anti-nodal planes dose to the side wads (FIG. 2, Panel C).The end of the separation channel is split into three or more outletchannels, thus allowing the positive Φ-factor particles to exit througha center outlet and the negative Φ-factor particles to exit through sideoutlets (FIG. 1).

An example of an embodiment in which an acoustophoresis device of thepresent disclosure is based upon the Lund-method is shown in FIG. 3,which provides a schematic cross-section of an acoustophoresis separatorchip. In this embodiment, the separator chip 100 includes a chip 12 inwhich a channel 10 has been formed, such as by etching. A membrane 14(e.g., a glass membrane, such as boron silica glass) is attached to thetop of the chip 12. A vibration transducer 20 is affixed to the bottomof the chip 12, using affixing means 18 (e.g., adhesives, gels, and thelike). In some embodiments, affixing means 18 may involve gluing thevibration transducer 20 to the chip 12, or using a fluid such asglycerol to couple the acoustic energy of the vibration transducer 20 tothe channel 10 contained within the chip 12.

Acoustophoresis devices, such as the separator chip 100 depicted in FIG.3, may be manufactured from any convenient material. In certainembodiments, the material has is a high Q-value material (i.e.,transmits sound waves at low losses) that displays good acousticreflection properties when the sound wave transits from the fluidicmedia into the boundary wall, and/or the material may offer a lowtemperature rise (e.g., silicon). In certain aspects, one or more flowchannels 10 are made by etching (e.g., anisotropically etching) achannel in silicon, glass (e.g., Pyrex glass), steel, Poly(methylmethacrylate), polycarbonate, or any other convenient material. Thechannel(s) may be sealed using a membrane 14 sealed atop the channel.Any convenient membrane type may be used, such as glass (e.g., boronsilica glass). In certain aspects, a vibration generator 20 is bonded tothe bottom of the channel, Vibration generators of interest include, butare not limited to, piezoelectric transducers such as PZT. In certainaspects, the piezoelectric transducer is of the multi-layer type, but abimorph piezoelectric element may also be used as well as any other kindof ultrasound generating element with suitable dimensions.

In certain aspects, the dimensions for a channel in which to performacoustic focusing are about 375 μm×about 150 μm×about 30-70 mm. In otheraspects, the channel may vary, for example from about 100-550 μm×about50-250 μm×about 20-100 mm. In certain aspects, the width of the channelmay be chosen to correspond to half the desired ultrasonic wavelength,thereby creating a resonator between the side walls of the flow channelin which a standing wave can be formed. The resonant frequency of thechannel may, in certain embodiments, depend on the width of the channel,the speed of sound in the liquid medium or media passing through thechip, and/or other factors known in the art.

In certain embodiments, the frequency of the acoustic wave that isapplied corresponds to the fundamental resonance mode of the vibrationtransducer (e.g., about 2 MHz for many PZT plates) and/or is dependentupon the resonant frequency of the channel (e.g., as described above).The frequency may, in some embodiments, correspond to a harmonic of thevibration transducer, such as a first harmonic, second harmonic, and thelike. In various aspects, the frequency applied may be about 1.5 MHz ormore, including about 1.9 MHz or more, e.g. about 2.0 MHz to about 2.15MHz or more, such as about 2.0 MHz to about 2.1 MHz, about 2.1 to about2.2 MHz, about 2.2 MHz to about 2.3 MHz, about 2.3 to about 2.4 MHz,about 2.5 MHz to about 3.0 MHz, about 3.0 MHz to about 3.5 MHz, about3.5 to about 4.0 MHz, about 4.0 MHz to about 5.0 MHz, or about 5.0 MHzto 10.0 MHz.

The activation voltage that is applied may also vary. For example, incertain aspects an activation voltage is about 0.1 V_(pp) to about 100V_(pp), such as about 0.1 V_(pp) to about 1 V_(pp), about 1 V_(pp) toabout 10 V_(pp), about 10 V_(pp) to about 20 V_(pp), about 20 V_(pp) toabout 30 V_(pp), about 30 V_(pp) to about 40 V_(pp), about 40 V_(pp) toabout 50 V_(pp), about 50 V_(pp) to about 75 V_(pp), about 75 V_(pp) toabout 100 V_(pp).

In certain embodiments, the acoustophoresis device may include one ormore cooling elements (e.g., a Peltier element, heatsink, heat pipe,cooling fan, and the like) to cool the vibration transducer and/or toavoid excessive heating of the therapeutic agents or media. For example,in certain aspects the activation voltage is about 25-30 30 V_(pp) orhigher, and the acoustophoresis device contains one or more coolingelements configured to keep the temperature of the vibration transducerbelow a given value (e.g., about 40° C. or less, such as about 37° C.).

In certain aspects, an acoustophoresis device may be controlled by aprocessor configured to control the vibration generator. The processormay be contained within a control unit or control box. In certainaspects, the processor is configured to control the vibration generatorby altering one or more of the shape, frequency and power of theelectrical energy delivered to the vibration generator.

The flow rate of an acoustophoresis device may vary. In certainembodiments, the flow rate of the acoustophoresis device is adjustedsuch that the output from the acoustophoresis device is optimal forsubsequent analysis, such as sorting on a flow cytometer. In otheraspects, the flow rate of the acoustophoresis device is adjusted suchthat the output from the acoustophoresis device facilitatesadministration of the therapeutic agent to a subject.

In certain aspects, the rate at which one or more acoustophoresisdevices exchange a therapeutic agent contained in a first medium into asecond medium is about 1 μl/min or more. For example, in certain aspectsthe rate is about 10 μl/min to 1 L/min, including about 10 μl/min toabout 50 μl/min, about 50 μl/min to about 100 μl/min, about 100 μl/minto about 200 μl/min, about 200 μl/min to about 300 μl/min, about 300μl/min to about 400 μl/min, about 400 μl/min to about 500 μl/min, about500 μl/min to about 600 μl/min, about 600 μl/min to about 700 μl/min,about 700 μl/min to about 800 μl/min, about 800 μl/min to about 900μl/min, about 900 μl/min to about 1 ml/min, about 1 ml/min to about 10ml/min, about 10 ml/min to about 20 ml/min, about 20 ml/min to about 30ml/min, about 30 ml/min to about 40 ml/min, about 40 ml/min to about 50ml/min, about 50 ml/min to about 60 ml/min, about 60 ml/min to about 70ml/min, about 70 ml/min to about 80 ml/min, about 80 ml/min to about 90ml/min, about 90 ml/min to about 100 ml/min, about 100 ml/min to about150 ml/min, about 150 ml/min to about 200 ml/min, about 200 ml/min toabout 500 ml/min, or about 500 ml/min to 1 L/min.

In certain aspects, the flow rate may be controlled by modulating one ormore pumps (e.g., a syringe pump, such as a WPI sp210iwz distributed byWorld Precision Instruments Inc., Sarasota, Fla.; elastomeric pumps;and/or peristaltic pumps) or valves (e.g., pinch valves). The flow ratemay, in certain embodiments, be controlled by a processor, such as aprocessor described above. In other aspects, the flow rate may bedetermined by gravimetric fluid flow.

In certain aspects, to achieve a desired flow rate a plurality ofparallel separation channels may be used in one or more acoustophoresisdevices. For example, in certain aspects two or more parallel separationchannels are used, including 3 or more, such as 5 or more, 8 or more, 15or more, 25 or more, 40 or more, 60 or more, 80 or more, 100 or more,125 or more, 150 or more, 200 or more, 300 or more, 400 or more, 500 ormore, or about 500 to 1000. The separation channels may be contained onone or more chips, such as 2 or more, 5 or more, 10 or more, 20 or more,50 or more, or about 50 to 100. Moreover, a plurality of vibrationtransducers may be used, such as 2 or more, 5 or more, or about 10 to50.

In certain aspects, two or more separation channels are arranged inseries. For example, FIG. 5 presents an embodiment in which the outputfrom one acoustophoresis device is used as an input to a secondacoustophoresis device. Any convenient number of acoustophoresis devicesand/or separation channels may be arranged in series and/or in parallelto facilitate the transfer of a therapeutic agent from a first mediuminto a second medium.

Accordingly, in some embodiments, the subject methods may involve theuse of two or more acoustophoresis devices, such as 3 or more, including4 or more, 5 or more, 6 or more, or 7 to 10. Such acoustophoresisdevices may be arranged in any convenient configuration, such as in aserial configuration, parallel configuration, or a combination of thetwo. Moreover, the acoustophoresis devices may be substantiallyidentical, identical, or heterogeneous (e.g., differ in one or moreways, such as in the dimensions of the flow channel, the appliedvoltage, the oscillation frequency, etc.).

Moreover, the acoustophoresis devices used in practicing the subjectmethods may in some aspects contain one or more additional components.Examples of such components include, but are not limited to, one or morevalves (e.g., pinch valves, and the like), reservoirs (e.g., samplereservoirs, wash reservoirs, waste reservoirs, and the like), pumps(e.g., syringe pumps, peristaltic pumps, and the like), connectivetubing (e.g., silicone tubing), housings, processors, and the like.

Administration of a Therapeutic Agent to a Subject

In certain aspects, the subject methods include administering thetherapeutic agent suspended in the second (or third, fourth, fifth,etc.) medium to a subject. The methods may involve administration of atherapeutic agent to a variety of subjects. In many embodiments thesubjects are “mammals” or “mammalian”, where these terms are usedbroadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the subjects are humans. The subjectmethods may be applied to human subjects of both genders and at anystage of development (i.e., neonates, infant, juvenile, adolescent,adult), where in certain embodiments the human subject is a juvenile,adolescent or adult. While the present invention may be applied to ahuman subject, it is to be understood that the subject methods may alsobe carried-out on other animal subjects (that is, in “non-humansubjects”) such as, but not limited to, birds, mice, rats, dogs, cats,livestock and horses.

FIG. 4 provides a schematic depiction of such an embodiment of thepresent invention. In this embodiment, a gravimetric fluid flow is usedto pass cells through the acoustophoresis device wherein cells aretransferred from a first storage medium into a second, sterilephysiological medium, labeled as a wash buffer. Alternatively, a pumpmechanism, such as a peristaltic pump, can be used to control the flowof the fluid through the acoustophoresis device and into a patient. Alltubing and the acoustophoresis chip can be connected and maintained inan entirely closed, aseptic system using connectors and methods known inthe art.

Any convenient means of administration may be used in practicing thesubject methods. Examples of routes of administration include, but arenot limited to, parenteral (e.g., subcutaneous, intramuscular,intradermal, intravenous and intrathecal), intraperitoneal,intravesicular, oral, etc., administration. In certain aspects, thesubject methods may be performed at a subject's bedside.

In certain aspects, the subject methods may involve allotransplantationand/or xenotransplantation. Aspects further include therapeutic agentsbeing obtained from a subject, manipulated and/or processed in one ormore ways, and re-administered to the subject.

Accordingly, in certain aspects, the subject methods are performed understerile conditions and/or are sterile. By “sterile” is meant a samplethat is free or substantially free from live bacteria or othermicroorganisms.

Devices

Also provided are devices for practicing the subject methods. In certainaspects, the devices may include one or more acoustophoresis devicesconfigured to connect with one or more containers. Embodiments of thedevices may include connectors configured to connect with one or morecontainers so as to maintain an entirely closed, aseptic system. Incertain aspects, the device may be configured to connect to a containercontaining a sterile physiologically compatible buffer and a containercontaining a therapeutic agent in, e.g., storage buffer or freezingbuffer.

Embodiments further include one or more one or more acoustophoresisdevices in fluidic communication with a container containing a sterilephysiologically compatible buffer, and configured to connect with acontainer containing a therapeutic agent (e.g., in storage buffer,freezing buffer, etc.). Such devices may be sterile. The devices mayinclude connectors configured to connect with the container containing atherapeutic agent so as to maintain an entirely closed, aseptic system.

In certain aspects, the devices include a first container providing thetherapeutic agent suspended in a first medium; a second containerproviding a second medium; an acoustophoresis device (e.g., as describedabove) in fluidic communication with the first and second containers andconfigured to exchange the second medium for the first medium, toproduce the therapeutic agent suspended in the second medium.

In certain aspects, devices may include more than two containers, suchas 3 or more, 4 or more, or 5 or more. The containers may contain athird medium, fourth medium, fifth medium, etc. Moreover, devices maycontain 2 or more acoustophoresis devices, such as 3 or more, including4 or more, 5 or more, 6 or more, e.g., 7 to 10. Such acoustophoresisdevices may be arranged in any convenient configuration, such as in aserial configuration, parallel configuration, or a combination of thetwo. Moreover, the acoustophoresis devices may be substantiallyidentical, identical, or heterogeneous (e.g., differ in one or moreways, such as in the dimensions of the flow channel, the appliedvoltage, the oscillation frequency, etc.). The containers andacoustophoresis devices may be fluidically coupled using tubing,connectors, and methods known in the art. The devices may be integratedinto the same article of manufacture as a single unit, or distributedamong two or more different units (e.g., as a system) where the two ormore different units are in communication with each other, e.g., via awired or wireless communication protocol.

Accordingly, aspects of the present disclosure further include systems,e.g., computer based systems, which are configured to transfertherapeutic agents as described above. A “computer-based system” refersto the hardware, software, and data storage devices used to analyze theinformation of the present invention. The minimum hardware ofembodiments of the computer-based systems includes a central processingunit (CPU) (e.g., a processor), an input device, an output device, anddata storage device. Any one of the currently available computer-basedsystems may be suitable for use in the embodiments disclosed herein. Thedata storage device may include any manufacture including a recording ofthe present information as described above, or a memory access meansthat can access such a manufacture. For example, embodiments of thesubject systems may include the following components: (a) acommunications module for facilitating information transfer between thesystem and one or more users, e.g., via a user computer or workstation;and (b) a processing module for performing one or more tasks involved inthe analysis of the magnetically labeled moieties.

In certain aspects, a system may operate in a closed-loop fashion. Forexample, in some embodiments a system may measure one or more parametersof the exchange of the therapeutic agent, such as the flow rate or therate of administration of the therapeutic agent to a subject, and thelike. The system may change one or more parameters of the subjectacoustophoresis devices on a substantially real-time basis toautomatically obtain the desired results. For example, the system mayalter one or more of the flow rate of an acoustophoresis device, thefrequency of the vibration generator of an acoustophoresis device, thepower applied to the vibration generator, etc. In certain aspects, sucha closed-loop system may involve applying one or more statistical orlearning machine algorithms, such as genetic algorithms, neuralnetworks, hidden Markov models, Bayesian networks, and the like.

Additionally, systems of the present disclosure may include a number ofadditional components, such as data output devices, e.g., monitors,printers, and/or speakers, data input devices, e.g., interface ports, akeyboard, a mouse, etc., fluid handling components (e.g., pumps, valves,and the like), power sources, etc.

Kits

Also provided are kits for practicing one or more embodiments of theabove-described methods. The subject kits may include various componentsand reagents.

In some instances, the kits include one or more acoustophoresis devicesconfigured to connect with one or more containers. The kits may includea container containing a sterile physiologically compatible buffer, andthe acoustophoresis device(s) may be configured to administertherapeutic agent transferred into the physiologically compatible bufferinto a subject. In some embodiments, the kits are configured to be usedat a subject's bedside, e.g., using gravimetric flow and/ormechanically-assisted fluid flow (e.g., as described above)

In some instances, the kits include at least reagents finding use in themethods (e.g., as described above); and a computer readable mediumhaving a computer program stored thereon, wherein the computer program,when loaded into a computer, operates the computer to performacoustophoresis as described herein; and a physical substrate having anaddress from which to obtain the computer program.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the methods. These instructions maybe present in the subject kits in a variety of forms, one or more ofwhich may be present in the kit. One form in which these instructionsmay be present is as printed information on a suitable medium orsubstrate, e.g., a piece or pieces of paper on which the information isprinted, in the packaging of the kit, in a package insert, etc. Yetanother means would be a computer readable medium, e.g., CD, DVD,Blu-Ray, flash memory, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the Internet to access the information at a removed site. Anyconvenient means may be present in the kits.

Utility

The subject methods, devices, and kits find use in a variety ofdifferent applications where it is desirable to exchange particles(e.g., therapeutic agents, such as cells) from one liquid medium toanother liquid medium.

For example, embodiments of the subject methods may facilitate thetransfer of a therapeutic agent from a storage medium (e.g., a mediumcontaining a cryoprotectant) to an infusion medium that may beadministered into a subject. The exchange of the therapeutic agent fromthe storage medium to the infusion medium thus may prevent complicationsor adverse reactions in the subject from exposure to one or morecomponents of the storage medium (e.g., the cryoprotectant). The subjectdevices and methods may be utilized at a subject's bedside and, incertain embodiments, may be performed under sterile conditions. Becausethe subject methods and devices may rely in some embodiments solely ongravimetric fluid flow, the methods and devices may be less expensiveand/or easier to administer than existing technologies.

NON-LIMITING EMBODIMENTS

Non-limiting exemplary embodiments of the present disclosure areprovided as follows:

-   -   1. A method of preparing a particulate infusion composition, the        method comprising:        -   combining a first liquid comprising a therapeutic agent in a            storage medium and a second liquid infusion medium in a            manner sufficient to produce a laminar flow of the first and            second media; and        -   acoustophoretically moving the therapeutic agent from a            first liquid medium into the second liquid medium.    -   2. The method according to 1, wherein the method further        comprises infusing the second liquid medium into a subject.    -   3. The method according to any of 1-2, wherein the storage        medium comprises a cryoprotectant.    -   4. The method according to 3, wherein the cryoprotectant is DMSO        or glycerol.    -   5. The method according to any of 2-4, wherein the method is        performed at the subject's bedside.    -   6. The method of any of 2-5, wherein the therapeutic agent was        obtained from the subject.    -   7. The method of any of 1-6, wherein the therapeutic agent is        contained within, associated with, or coupled to a particle to        facilitate acoustophoresis of the therapeutic agent.    -   8. The method of 7, wherein the particle is a liposome.    -   9. The method of 7, wherein the particle is a polymeric bead.    -   10. The method of any of 7-9, wherein the therapeutic agent is a        drug.    -   11. The method of any of 7-9, wherein the therapeutic agent is a        therapeutic protein.    -   12. The method of any of 7-9, wherein the therapeutic agent is a        nucleic acid.    -   13. The method of any of 1-7, wherein the therapeutic agent        comprises cells.    -   14. The method of 13, wherein the cells are peripheral blood        stem cells (PBSC), umbilical cord blood cells, hematopoietic        stem cells, or induced pluripotent stem cells.    -   15. The method of any of 1-14, wherein combining the first and        second media comprises gravimetric fluid flow.    -   16. The method of any of 1-15, wherein combining the first and        second media comprises mechanically-assisted fluid flow.    -   17. The method of any of 1-16, further comprising combining the        second liquid medium with a third liquid medium in a manner        sufficient to produce a laminar flow of the second and third        media; and        -   acoustophoretically moving the therapeutic agent from the            second liquid medium into the third liquid medium.    -   18. The method of 17, comprising administering the therapeutic        agent suspended in the third medium to a subject.    -   19. The method of 18, wherein said administering comprises        parenteral, intraperitoneal, or intravesicular administration.    -   20. The method of any of 18-19, wherein the subject is        mammalian.    -   21. The method of 20, wherein the subject is human.    -   22. The method of any of 1-21, wherein the method is sterile.    -   23. A device for transferring a therapeutic agent, the device        comprising:        -   a first container providing the therapeutic agent suspended            in a first medium;        -   a second container providing a second medium;        -   an acoustophoresis device in fluidic communication with the            first and second containers and configured to exchange the            second medium for the first medium, to produce the            therapeutic agent suspended in the second medium.    -   24. The device of 23, further comprising a channel in fluidic        communication with the acoustophoresis device and configured to        receive the therapeutic agent suspended in the second medium and        administer the therapeutic agent suspended in the second medium        to a patient.    -   25. The device of 23 or 24, comprising at least one pump to        control a flow rate of the first medium or the second medium        through the acoustophoresis device.    -   26. The device of any of 23-25, wherein the acoustophoresis        device comprises 2 or more separation channels.    -   27. The device of any of 23-26, wherein the acoustophoresis        device comprises 5 or more separation channels.    -   28. The device of any of 23-27, wherein the acoustophoresis        device comprises 10 or more separation channels.    -   29. The device of any of 23-28, wherein the acoustophoresis        device exchanges the second medium for the first medium, to        produce the therapeutic agent suspended in the second medium, at        a rate of about 1 μl/min or more.    -   30. The device of any of 23-29, wherein the acoustophoresis        device exchanges the second medium for the first medium, to        produce the therapeutic agent suspended in the second medium, at        a rate of about 100 μl/min or more.    -   31. The device of any of 23-30, wherein the acoustophoresis        device exchanges the second medium for the first medium, to        produce the therapeutic agent suspended in the second medium, at        a rate of about 1 ml/min or more.    -   32. The device of any of 23-31, wherein the acoustophoresis        device exchanges the second medium for the first medium, to        produce the therapeutic agent suspended in the second medium, at        a rate of about 100 ml/min or more.    -   33. The device of any of 23-32, wherein the acoustophoresis        device comprises a separation chip made of silicon, steel,        glass, Poly(methyl methacrylate), or polycarbonate.    -   34. The device of any of 23-33, wherein the acoustophoresis        device comprises a piezoceramic transducer.    -   35. The device of any of 23-34, wherein the device is sterile.    -   36. The device of any of 23-35, wherein the therapeutic agent        was obtained from the subject.    -   37. The device of any of 23-36, wherein the therapeutic agent        comprises cells.    -   38. The device of 37, wherein the cells are peripheral blood        stem cells (PBSC), umbilical cord blood cells, hematopoietic        stem cells, or induced pluripotent stem cells.    -   39. The device of any of 23-38, wherein the device comprises a        processor configured to control the exchange the second medium        for the first medium in the acoustophoresis device.    -   40. The device of 39, wherein the processor is configured to        control the device under a closed-loop feedback mechanism.    -   41. A kit comprising:        -   an acoustophoresis device configured to connect with two or            more containers; and        -   a container providing a sterile physiologically compatible            buffer.    -   42. The kit of 41, wherein the kits is configured to be used at        a subject's bedside.    -   43. An acoustophoresis device configured to connect with a first        container comprising a therapeutic agent in a storage medium and        a second container comprising infusion medium; and configured to        acoustophoretically move the therapeutic agent from the medium        into the infusion medium.

EXAMPLES

As can be appreciated from the disclosure provided above, the presentdisclosure has a wide variety of applications. Accordingly, thefollowing examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Those of skill in the art will readily recognizea variety of noncritical parameters that could be changed or modified toyield essentially similar results. Thus, the following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the presentinvention, and are not intended to limit the scope of what the inventorsregard as their invention nor are they intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (e.g.amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for.

Acoustophoresis chips were fabricated as described in U.S. Pat. No.6,929,750; Laurell, et al. (2007) Chem. Soc. Rev., 2007, 36, 492-506;Petersson, et al. (2005) Analytical Chemistry 77: 1216-1221; andAugustsson, et al. (2009) Lab on a Chip 9: 810-818; the disclosures ofwhich are incorporated herein by reference.

The initial chip size was reduced about 10 fold (from about 15×75 mm to3×35 mm) with accompanying 10× reductions in power consumption and cost,while performing equivalently to the larger precursors. That is, FACSlysed whole blood, ammonium chloride lysed whole blood, and PBMC's fromBD Vacutainer® CPT™ Cell Preparation Tubes could be washed with >90%recovery of lymphocytes and 95% rejection of debris and small moleculecontamination.

The acoustophoresis chip was integrated into a holder with standardizedfluidic connections and shown to function correctly to 35 psi withoutleakage. Configuration and attachment of the PZT driver was alsoinvestigated and optimized so that the chip and driver could beintegrated as a finished assembly into the flow path of a cytometer in asimple, reproducible manner.

This assembly was interfaced to a modified BD FACSCanto™ analyticalcytometer. Pressure driven fluidics were developed to supply wash bufferto the chip. Use of the washer on FACS lysed blood was investigated indetail on the BD FACSCanto™ analytical cytometer by testing multiplesamples on multiple days. Samples were processed in lyse-no-wash (LNW),lyse-wash (LW) and chip-wash (CW) formats. BD TruCount™ control beadrecovery was used to normalize results.

WBC recovery was 104±5% for LW and 98±6% for CW relative to LNW control.WBC subpopulations appeared unchanged relative to the LNW control.Debris removal for LW and CW methods was 99.6±0.2% and 98.5±0.9%respectively. CW and LW formats completely resolved the dimly labeled BCell population from background.

The acoustophoresis chip was also interfaced to the BD Influx™ flowcytometer and successfully used to wash PBMC samples from BD Vacutainer®CPT™ Cell Preparation Tubes. The sorter was programmed and shown torecover only lymphocytes and remove all other monocytes and BD TruCount™control beads.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this disclosure that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention being withoutlimitation to such specifically recited examples and conditions.Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of present invention is embodied bythe appended claims.

What is claimed is:
 1. A device for transferring a therapeutic agentfrom a first liquid to a second liquid infusion medium, the devicecomprising: a) a first container comprising a first liquid comprising atherapeutic agent suspended in a first medium comprising acryoprotectant agent; b) second container comprising a second liquidinfusion medium; and c) an acoustophoresis device in fluidiccommunication with the first and second containers and configured toacoustophoretically move the therapeutic agent from the first liquidinto the second liquid infusion medium, to produce the therapeutic agentsuspended in the second liquid infusion medium; wherein the device issterile.
 2. The device according to claim 1, further comprising achannel in fluidic communication with the acoustophoresis device andconfigured to receive the therapeutic agent suspended in the secondliquid infusion medium and administer the therapeutic agent suspended inthe second medium to a patient.
 3. The device according to claim 2,comprising at least one pump to control a flow rate of the first mediumor the second medium through the acoustophoresis device.
 4. The deviceaccording to claim 2, wherein the acoustophoresis device comprises 2 ormore separation channels.
 5. The device according to claim 1, whereinthe first container comprises a freezing bag.
 6. The device according toclaim 1, wherein the acoustophoresis device further comprises: d) achannel having a central inlet configured to receive the second liquidinfusion medium, e) two side inlets configured to receive the firstliquid comprising the therapeutic agent, f) a central outlet configuredto output a liquid comprising the therapeutic agent in the second liquidinfusion medium, and g) a waste liquid outlet.
 7. The device accordingto claim 1, comprising at least one pump to control a flow rate of thefirst liquid or the second liquid infusion medium through theacoustophoresis device.
 8. The device according to claim 7, wherein theacoustophoresis device comprises 5 or more separation channels.
 9. Thedevice according to claim 8, wherein the acoustophoresis devicecomprises 10 or more separation channels.
 10. The device according toclaim 1, wherein the acoustophoresis device comprises a separation chipmade of a material selected from the group consisting of silicon, steel,glass, Poly(methyl methacrylate) and polycarbonate.
 11. The deviceaccording to claim 1, wherein the acoustophoresis device comprises apiezoceramic transducer.
 12. The device according to claim 1, whereinthe therapeutic agent comprises cells.
 13. The device according to claim12, wherein the cells are peripheral blood stem cells (PBSC), umbilicalcord blood cells, hematopoietic stem cells, or induced pluripotent stemcells.
 14. The device according to claim 1, wherein the device comprisesa processor configured to control the movement of the therapeutic agentfrom the first liquid to the second liquid storage medium in theacoustophoresis device.
 15. The device according to claim 14, whereinthe processor is configured to control the device under a closed-loopfeedback mechanism.
 16. A device for transferring a therapeutic agentfrom a first liquid to a second liquid infusion medium, the devicecomprising: a) a freezing bag comprising the first liquid comprising thetherapeutic agent suspended in a first medium comprising acryoprotectant agent, wherein the therapeutic agent comprises cells; b)second container comprising the second liquid infusion medium; c) anacoustophoresis device in fluidic communication with the first andsecond containers and configured to acoustophoretically move thetherapeutic agent from the first liquid into the second liquid infusionmedium to produce the therapeutic agent suspended in the second liquidinfusion medium; wherein the device is sterile.
 17. The device accordingto claim 16, further comprising a channel in fluidic communication withthe acoustophoresis device and configured to receive the therapeuticagent suspended in the second liquid infusion medium and administer thetherapeutic agent suspended in the second medium to a patient.
 18. Thedevice according to claim 17, comprising at least one pump to control aflow rate of the first medium or the second medium through theacoustophoresis device.
 19. The device according to claim 18, whereinthe acoustophoresis device comprises 2 or more separation channels. 20.The device according to claim 16, wherein the device comprises aprocessor configured to control the movement of the therapeutic agentfrom the first liquid to the second liquid storage medium in theacoustophoresis device.